Added Value of Deep Sequencing Relative to Population Sequencing in Heavily Pre-Treated HIV-1-Infected Subjects.

OBJECTIVE: To explore the potential of deep HIV-1 sequencing for adding clinically relevant information relative to viral population sequencing in heavily pre-treated HIV-1-infected subjects. METHODS: In a proof-of-concept study, deep sequencing was compared to population sequencing in HIV-1-infected individuals with previous triple-class virological failure who also developed virologic failure to deep salvage therapy including, at least, darunavir, tipranavir, etravirine or raltegravir. Viral susceptibility was inferred before salvage therapy initiation and at virological failure using deep and population sequencing genotypes interpreted with the HIVdb, Rega and ANRS algorithms. The threshold level for mutant detection with deep sequencing was 1%. RESULTS: 7 subjects with previous exposure to a median of 15 antiretrovirals during a median of 13 years were included. Deep salvage therapy included darunavir, tipranavir, etravirine or raltegravir in 4, 2, 2 and 5 subjects, respectively. Self-reported treatment adherence was adequate in 4 and partial in 2; one individual underwent treatment interruption during follow-up. Deep sequencing detected all mutations found by population sequencing and identified additional resistance mutations in all but one individual, predominantly after virological failure to deep salvage therapy. Additional genotypic information led to consistent decreases in predicted susceptibility to etravirine, efavirenz, nucleoside reverse transcriptase inhibitors and indinavir in 2, 1, 2 and 1 subject, respectively. Deep sequencing data did not consistently modify the susceptibility predictions achieved with population sequencing for darunavir, tipranavir or raltegravir. CONCLUSIONS: In this subset of heavily pre-treated individuals, deep sequencing improved the assessment of genotypic resistance to etravirine, but did not consistently provide additional information on darunavir, tipranavir or raltegravir susceptibility. These data may inform the design of future studies addressing the clinical value of minority drug-resistant variants in treatment-experienced subjects.

Failure to demonstrate experimental vertical transmission of the epidemic strain of Chikungunya virus in Aedes albopictus from La Réunion Island, Indian Ocean

Aedes albopictus was responsible for transmission in the first outbreak of chikungunya (CHIK) on La Réunion Island, Indian Ocean, in 2005-2006. The magnitude of the outbreak on this island, which had been free of arboviral diseases for over 30 years, as well as the efficiency of Ae. albopictus as the main vector, raises questions about the maintenance of the CHIK virus (CHIKV) through vertical transmission mechanisms. Few specimens collected from the field as larvae were found to be infected. In this study, Ae. albopictus originating from La Réunion were orally infected with a blood-meal containing 10(8) pfu/mL of the CHIKV epidemic strain (CHIKV 06.21). Eggs from the first and second gonotrophic cycles were collected and raised to the adult stage. The infectious status of the progeny was checked (i) by immunofluorescence on head squashes of individual mosquitoes to detect the presence of viral particles or (ii) by quantitative RT-PCR on mosquito pools to detect viral RNA. We analysed a total of 1,675 specimens from the first gonotrophic cycle and 1,709 from the second gonotrophic cycle without detecting any viral particles or viral RNA. These laboratory results are compared to field records.

Assessment of adipose tissue metabolism by means of subcutaneous microdialysis in patients with sepsis or circulatory failure.

To evaluate the role of adipose tissue in the metabolic stress response of critically ill patients, the release of glycerol and lactate by subcutaneous adipose tissue was assessed by means of microdialysis in patients with sepsis or circulatory failure and in healthy subjects. Patients with sepsis had lower plasma free fatty acid concentrations and non-significant elevations of plasma glycerol concentrations, but higher adipose-systemic glycerol concentrations gradients than healthy subjects or patients with circulatory failure, indicating a stimulation of subcutaneous adipose lipolysis. They also had a higher lipid oxidation. Lipid metabolism (adipose-systemic glycerol gradients, lipid oxidation) was not altered in patients with circulatory failure. These observations highlight major differences in lipolysis and lipid utilization between patients with sepsis and circulatory failure. Hyperlactataemia was present in both groups of patients, but the adipose-systemic lactate concentration gradient was not increased, indicating that lactate production by adipose tissue was not involved. This speaks against a role of adipose tissue in the development of hyperlactataemia in critically ill patients.

Consensus statement of the European guidelines on clinical management of HIV-1 tropism testing

Introduction: Testing for HIV tropism is recommended before prescribing a chemokine receptor blocker. To date, in most European countries HIV tropism is determined using a phenotypic test. Recently, new data have emerged supporting the use of a genotypic HIV V3-loop sequence analysis as the basis for tropism determination. The European guidelines group on clinical management of HIV-1 tropism testing was established to make recommendations to clinicians and virologists. Methods: We searched online databases for articles from Jan 2006 until March 2010 with the terms: tropism or CCR5-antagonist or CCR5 antagonist or maraviroc or vicriviroc. Additional articles and/or conference abstracts were identified by hand searching. This strategy identified 712 potential articles and 1240 abstracts. All were reviewed and finally 57 papers and 42 abstracts were included and used by the panel to reach a consensus statement. Results: The panel recommends HIV-tropism testing for the following indications: i) drug-naïve patients in whom toxicity or limited therapeutic options are foreseen; ii) patients experiencing therapy failure whenever a treatment change is considered. Both the phenotypic Enhanced Trofile assay (ESTA) and genotypic population sequencing of the V3-loop are recommended for use in clinical practice. Although the panel does not recommend one methodology over another it is anticipated that genotypic testing will be used more frequently because of its greater accessibility, lower cost and shorter turnaround time. The panel also provides guidance on technical aspects and interpretation issues. If using genotypic methods, triplicate PCR amplification and sequencing testing is advised using the G2P interpretation tool (clonal model) with an FPR of 10%. If the viral load is below the level of reliable amplification, proviral DNA can be used, and the panel recommends performing triplicate testing and use of an FPR of 10%. If genotypic DNA testing is not performed in triplicate the FPR should be increased to 20%. Conclusions: The European guidelines on clinical management of HIV-1 tropism testing provide an overview of current literature, evidence-based recommendations for the clinical use of tropism testing and expert guidance on unresolved issues and current developments. Current data support both the use of genotypic population sequencing and ESTA for co-receptor tropism determination. For practical reasons genotypic population sequencing is the preferred method in Europe.

Validity and reliability of the Spanish version of the DN4 (Douleur Neuropathique 4 questions) questionnaire for differential diagnosis of pain syndromes associated to a neuropathic or somatic component

BACKGROUND This study assesses the validity and reliability of the Spanish version of DN4 questionnaire as a tool for differential diagnosis of pain syndromes associated to a neuropathic (NP) or somatic component (non-neuropathic pain, NNP). METHODS A study was conducted consisting of two phases: cultural adaptation into the Spanish language by means of conceptual equivalence, including forward and backward translations in duplicate and cognitive debriefing, and testing of psychometric properties in patients with NP (peripheral, central and mixed) and NNP. The analysis of psychometric properties included reliability (internal consistency, inter-rater agreement and test-retest reliability) and validity (ROC curve analysis, agreement with the reference diagnosis and determination of sensitivity, specificity, and positive and negative predictive values in different subsamples according to type of NP). RESULTS A sample of 164 subjects (99 women, 60.4%; age: 60.4 +/- 16.0 years), 94 (57.3%) with NP (36 with peripheral, 32 with central, and 26 with mixed pain) and 70 with NNP was enrolled. The questionnaire was reliable [Cronbach's alpha coefficient: 0.71, inter-rater agreement coefficient: 0.80 (0.71-0.89), and test-retest intra-class correlation coefficient: 0.95 (0.92-0.97)] and valid for a cut-off value > or = 4 points, which was the best value to discriminate between NP and NNP subjects. DISCUSSION This study, representing the first validation of the DN4 questionnaire into another language different than the original, not only supported its high discriminatory value for identification of neuropathic pain, but also provided supplemental psychometric validation (i.e. test-retest reliability, influence of educational level and pain intensity) and showed its validity in mixed pain syndromes.

The Presence of Precursors of Benign Pre-B Lymphoblasts (Hematogones) in the Bone Marrow of a Paediatric Patient with Cytomegalovirus Infection

Hematogones are normal B-lymphoid precursors that multiply in the bone marrow of small children and of adults with ferropenic anaemia, neuroblastoma or idiopathic thrombocytopenic purpura. They are not normally found in peripheral blood, and the immunophenotype is virtually indistinguishable from that of B lymphoblasts. We discuss the case of a 3-month infant with an active cytomegalovirus infection, with hepatitis and pancytopenia associated with 13% hematogones in the bone marrow

Evaluating the probability and the impact of a failure in GMPLS based networks

In this paper, we define a new scheme to develop and evaluate protection strategies for building reliable GMPLS networks. This is based on what we have called the network protection degree (NPD). The NPD consists of an a priori evaluation, the failure sensibility degree (FSD), which provides the failure probability, and an a posteriori evaluation, the failure impact degree (FID), which determines the impact on the network in case of failure, in terms of packet loss and recovery time. Having mathematical formulated these components, experimental results demonstrate the benefits of the utilization of the NPD, when used to enhance some current QoS routing algorithms in order to offer a certain degree of protection

Intracoronary injection of bone marrow-derived mononuclear cells early or late after acute myocardial infarction: effects on global left ventricular function.

BACKGROUND: Intracoronary administration of autologous bone marrow-derived mononuclear cells (BM-MNC) may improve remodeling of the left ventricle (LV) after acute myocardial infarction. The optimal time point of administration of BM-MNC is still uncertain and has rarely been addressed prospectively in randomized clinical trials. METHODS AND RESULTS: In a multicenter study, we randomized 200 patients with large, successfully reperfused ST-segment elevation myocardial infarction in a 1:1:1 pattern into an open-labeled control and 2 BM-MNC treatment groups. In the BM-MNC groups, cells were administered either early (ie, 5 to 7 days) or late (ie, 3 to 4 weeks) after acute myocardial infarction. Cardiac magnetic resonance imaging was performed at baseline and after 4 months. The primary end point was the change from baseline to 4 months in global LV ejection fraction between the 2 treatment groups and the control group. The absolute change in LV ejection fraction from baseline to 4 months was -0.4±8.8% (mean±SD; P=0.74 versus baseline) in the control group, 1.8±8.4% (P=0.12 versus baseline) in the early group, and 0.8±7.6% (P=0.45 versus baseline) in the late group. The treatment effect of BM-MNC as estimated by ANCOVA was 1.25 (95% confidence interval, -1.83 to 4.32; P=0.42) for the early therapy group and 0.55 (95% confidence interval, -2.61 to 3.71; P=0.73) for the late therapy group. CONCLUSIONS: Among patients with ST-segment elevation myocardial infarction and LV dysfunction after successful reperfusion, intracoronary infusion of BM-MNC at either 5 to 7 days or 3 to 4 weeks after acute myocardial infarction did not improve LV function at 4-month follow-up. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT00355186.

A MODEL OF CREATINE DEFICIENCY SYNDROMES IN 3D BRAIN CELL CULTURES BY KNOCKDOWN OF GAMT AND SLC6A8 GENES

La créatine joue un rôle essentiel dans le métabolisme cellulaire par sa conversion, par la creatine kinase, en phosphocreatine permettant la régénération de l'ATP. La synthèse de créatine, chez les mammifères, s'effectue par une réaction en deux étapes impliquant Γ arginine: glycine amidinotransférase (AGAT) et la guanidinoacétate méthyltransférase (GAMT). L'entrée de créatine dans les cellules s'effectue par son transporteur, SLC6A8. Les déficiences en créatine, dues au déficit en GAMT, AGAT ou SLC6A8, sont fréquentes et caractérisées par une absence ou une forte baisse de créatine dans le système nerveux central. Alors qu'il est connu que AGAT, GAMT et SLC6A8 sont exprimés par le cerveau, les conséquences des déficiences en créatine sur les cellules nerveuses sont peu comprises. Le but de ce travail était de développer de nouveaux modèles expérimentaux des déficiences en Cr dans des cultures 3D de cellules nerveuses de rat en agrégats au moyen de l'interférence à l'ARN appliquée aux gènes GAMT et SLC6A8. Des séquences interférentes (shRNAs) pour les gènes GAMT et SLC6A8 ont été transduites par des vecteurs viraux AAV (virus adéno-associés), dans les cellules nerveuses en agrégats. Nous avons ainsi démontré une baisse de l'expression de GAMT au niveau protéique (mesuré par western blot), et ARN messager (mesuré par qPCR) ainsi qu'une variation caractérisitique de créatine et guanidinoacétate (mesuré par spectrométrie de masse). Après avoir validé nos modèles, nous avons montré que les knockdown de GAMT ou SLC6A8 affectent le développement des astrocytes et des neurones ou des oligodendrocytes et des astrocytes, respectivement, ainsi qu'une augmentation de la mort cellulaire et des modifications dans le pattern d'activation des voies de signalisation impliquant caspase 3 et p38 MAPK, ayant un rôle dans le processus d'apoptose. - Creatine plays essential roles in energy metabolism by the interconversion, by creatine kinase, to its phosphorylated analogue, phosphocreatine, allowing the regeneration of ATP. Creatine is synthesized in mammals by a two step mechanism involving arginine:glycine amidinotransferase (AGAT) and guanidinoacetate methyltransferase (GAMT). Creatine is taken up by cells by a specific transporter, SLC6A8. Creatine deficiency syndromes, due to defects in GAMT, AGAT and SLC6A8, are among the most frequent inborn errors of metabolism, and are characterized by an absence or a severe decrease of creatine in central nervous system, which is the main tissue affected. While it is known that AGAT, GAMT and SLC6A8 are expressed in CNS, many questions remain on the specific effects of AGAT, GAMT and SLC6A8 deficiencies on brain cells. Our aim was to develop new experimental models of creatine deficiencies by knockdown of GAMT and SLC6A8 genes by RNAi in 3D organotypic rat brain cell cultures in aggregates. Specific shRNAs for the GAMT and SLC6A8 genes were transduced in brain cell aggregates by adeno-associated viruses (AAV). The AAV-transduced shRNAs were able to efficiently knockdown the expression of our genes of interest, as shown by a strong decrease of protein by western blotting, a decrease of mRNA by qPCR or characteristic variations of creatine and guanidinoacetate by tandem mass spectrometry. After having validated our experimental models, we have also shown that GAMT and SLC6A8 knockdown affected the development of astrocytes and neurons or oligodendrocytes and astrocytes, respectively. We also observed an increase of cell death and variations in activation pattern of caspase 3 and p38 MAPK pathways, involved in apoptosis, in our experimental model.

Tumor microenvironment and immune effects of antineoplastic therapy in lymphoproliferative syndromes.

Lymphomas represent a wide group of heterogenic diseases with different biological and clinical behavior. The underlying microenvironment-specific composition seems to play an essential role in this scenario, harboring the ability to develop successful immune responses or, on the contrary, leading to immune evasion and even promotion of tumor growth. Depending on surrounding lymphoid infiltrates, lymphomas may have different prognosis. Moreover, recent evidences have emerged that confer a significant impact of main lymphoma's treatment over microenvironment, with clinical consequences. In this review, we summarize these concepts from a pathological and clinical perspective. Also, the state of the art of lymphoma's anti-idiotype vaccine development is revised, highlighting the situations where this strategy has proven to be successful and eventual clues to obtain better results in the future.

Complete longitudinal analyses of the randomized, placebo-controlled, phase III trial of sunitinib in patients with gastrointestinal stromal tumor following imatinib failure.

PURPOSE: To analyze final long-term survival and clinical outcomes from the randomized phase III study of sunitinib in gastrointestinal stromal tumor patients after imatinib failure; to assess correlative angiogenesis biomarkers with patient outcomes. EXPERIMENTAL DESIGN: Blinded sunitinib or placebo was given daily on a 4-week-on/2-week-off treatment schedule. Placebo-assigned patients could cross over to sunitinib at disease progression/study unblinding. Overall survival (OS) was analyzed using conventional statistical methods and the rank-preserving structural failure time (RPSFT) method to explore cross-over impact. Circulating levels of angiogenesis biomarkers were analyzed. RESULTS: In total, 243 patients were randomized to receive sunitinib and 118 to placebo, 103 of whom crossed over to open-label sunitinib. Conventional statistical analysis showed that OS converged in the sunitinib and placebo arms (median 72.7 vs. 64.9 weeks; HR, 0.876; P = 0.306) as expected, given the cross-over design. RPSFT analysis estimated median OS for placebo of 39.0 weeks (HR, 0.505, 95% CI, 0.262-1.134; P = 0.306). No new safety concerns emerged with extended sunitinib treatment. No consistent associations were found between the pharmacodynamics of angiogenesis-related plasma proteins during sunitinib treatment and clinical outcome. CONCLUSIONS: The cross-over design provided evidence of sunitinib clinical benefit based on prolonged time to tumor progression during the double-blind phase of this trial. As expected, following cross-over, there was no statistical difference in OS. RPSFT analysis modeled the absence of cross-over, estimating a substantial sunitinib OS benefit relative to placebo. Long-term sunitinib treatment was tolerated without new adverse events.

Inflammatory Markers and Incident Heart Failure Risk in Older Adults: The Health, Aging, and Body Composition Study

Background: Inflammation is associated with heart failure (HF) risk factors and also directly affects myocardial function. However, the association between inflammation and HF risk in older adults has not been adequately evaluated. Methods: The association of baseline serum concentrations of interleukin-6 (IL-6), tumor necrosis factor alpha (TNF- ), and C-reactive protein (CRP) with incident HF was assessed with Cox proportional hazards models among 2610 older persons without prevalent HF enrolled in the Health, Aging, and Body Composition (Health ABC) Study (age, 73.6±2.9 years; 48.3% men; 59.6% white). Results: Median (interquartile range) baseline concentrations of IL-6, TNF- , and CRP were 1.80 (1.23, 2.76) pg/mL, 3.14 (2.41, 4.06) pg/mL, and 1.64 (0.99, 3.04) µg/mL, respectively. On follow-up (median, 9.4 years), 311 participants (11.9%) developed HF. In models controlling for clinical predictors of HF and incident coronary heart disease, doubling of IL-6, TNF- , and CRP concentrations was associated with 34% (95% CI, 18 -52%; P<.001), 33% (95% CI, 9 - 63%; P=.006), and 13% (95% CI, 3-24%; P=.01) increase in HF risk, respectively. In models including all 3 markers, IL-6 and TNF- , but not CRP, remained significant. Findings were similar across sex and race. Post-HF ejection fraction (EF) was available in 239 (76.8%) cases. When only cases with preserved EF were considered (n=105), IL-6 (HR per doubling, 1.57; 95% CI, 1.28 -1.94; P<.001), TNF- (HR per doubling, 1.59; 95% CI, 1.12-2.26; P=.01), and CRP (HR per doubling, 1.23; 95% CI, 1.05-1.44; P=.01) were all associated with HF risk in adjusted models. In contrast, when only cases with reduced EF (n=134) were considered, only IL-6 attained marginal significance in adjusted models (HR per doubling, 1.20; 95% CI, 0.99 -1.46; P=.06). Participants with 2 or 3 markers above median had pronounced HF risk in adjusted models (HR, 1.66; 95% CI, 1.12-2.46; P=.01; and HR, 1.76; 95% CI, 1.16 -2.65; P=.007, respectively). Addition of IL-6 to the clinical Health ABC HF model improved discrimination (C index from 0.717 to 0.734; P=.001) and fit (decreased Bayes information criterion by 17.8; P<.001). Conclusions: Inflammatory markers are associated with HF risk among older adults and may improve HF risk stratification.

Guidelines for specialized nutritional and metabolic support in the critically-ill patient. Update. Consensus SEMICYUC-SENPE: Acute renal failure

Nutritional support in acute renal failure must take into account the patient's catabolism and the treatment of the renal failure. Hypermetabolic failure is common in these patients, requiring continuous renal replacement therapy or daily hemodialysis. In patients with normal catabolism (urea nitrogen below 10 g/day) and preserved diuresis, conservative treatment can be attempted. In these patients, relatively hypoproteic nutritional support is essential, using proteins with high biological value and limiting fluid and electrolyte intake according to the patient's individual requirements. Micronutrient intake should be adjusted, the only buffering agent used being bicarbonate. Limitations on fluid, electrolyte and nitrogen intake no longer apply when extrarenal clearance techniques are used but intake of these substances should be modified according to the type of clearance. Depending on their hemofiltration flow, continuous renal replacement systems require high daily nitrogen intake, which can sometimes reach 2.5 g protein/kg. The amount of volume replacement can induce energy overload and therefore the use of glucose-free replacement fluids and glucose-free dialysis or a glucose concentration of 1 g/L, with bicarbonate as a buffer, is recommended. Monitoring of electrolyte levels (especially those of phosphorus, potassium and magnesium) and of micronutrients is essential and administration of these substances should be individually-tailored.

Acute coronary syndromes or myocarditis in young people: a difficult daily diagnostic challenge

Background: Chest pain (CP) represents about 5% of admissions to emergency departments (ED), even in young people. Acute coronary syndrome (ACS) and myocarditis are among the most important diagnoses to rule out. Clinical and ECG findings are not specific for either condition and separating both diagnoses is a challenge. Aim of the study: To evaluate the prevalence of ACS and myocarditis in young patients presenting with CP and elevated cardiac biomarkers to the ED and to determinate the differences in their clinical presentation. Methods: Retrospective study of all consecutive patients < 40 years old admitted to our ED from January 2009 to June 2011 for CP with elevated serum troponin concentration. All clinical, angiographic and cardiac magnetic resonance (CMR) data from the local database was reviewed. Clinical follow-up was obtained to assess all cause mortality, myocardial infarction and re-hospitalisation for CP. Results: 1588 patients < 40 years old were admitted to the ED with chest pain. 49 (3%) patients presenting with an elevated troponin I (> 0.09ug/l) were included in the study. 32.7% (16/49) were diagnosed with ACS (11 STEMI and 5 NSTEMI) and 59.2% (29/49) with myocarditis. Among the 29 patients with myocarditis, 17 presented with typical subepicardial late enhancement on CMR and 12 were diagnosed based on clinical presentation (6 had no complementary workup, 3 normal coronary angiogram and 3 inconclusive CMR). 8.1% (4/49) of patients had other diagnoses. Compared to patients with myocarditis, ACS patients were older (34.1±3.9 vs 26.9±6.4, p=0.0002) with significantly more cardiovascular risk factors (mean 2.06 vs 0.69, p<0.0001). Diabetes (18.8% vs 0%, p=0.004), dyslipidemia (56.3% vs 3.4%, p=0.0001) and family history of coronary artery disease (CAD) (37.5% vs 10.3%, p=0.050) were significantly associated with ACS. No significant association was found for smoking, hypertension and obesity. Fever (>38°C) or recent viral illness were present in 75.9% (22/29) of patients with myocarditis, and in 0% of ACS patients. During follow-up (mean 19.9 months ± 8.6), only 2 patients with myocarditis were re-admitted for chest pain. Conclusions: In this study, 32.7% of patients < 40 year old admitted to an ED with CP and elevated troponin had an ACS. Key clinical factors include diabetes, dyslipidemia, family history of CAD, fever or recent viral illness, and may help to differentiate ACS from myocarditis.

Influence of low-protein dietetic foods consumption on quality of life and levels of B vitamins and homocysteine in patients with chronic renal failure.

OBJECTIVE The aim of the study was to determine whether the consumption of low protein dietetic foods improved the quality of life and nutritional status for vitamins B and homocysteine in patients with chronic renal failure. METHODOLOGY This nutritional-intervention involved 28 men and 21 women, divided into two groups. The control-group consumed a low-protein diet prescribed, and the experimental-group consumed a diet in which some commonly used foods were replaced by low-protein dietetic foods. The study lasted 6 months. Food consumption was assessed by 24-h recall. Vitamin B6 as alphaEAST was measured in blood. Creatinine, urea, vitamin B12, folate and homocysteine were measured in plasma. The impact on the patients' quality of life from consuming the dietetic foods was assessed via the SF-36 questionnaire. RESULTS After 6 months, the protein intake among the experimental-group had decreased by 40%, and the urea/creatinine ratio and alphaEAST activity were also lower. The results of the SF-36 questionnaire show that the patients in the experimental-group obtained higher scores in the categories of general health and physical status. CONCLUSIONS The dietetic foods were very well accepted by all patients and their use allowed a better control of the protein intake, improved B6 status and a better quality of life.